A variety of techniques have been developed for separating, isolating, enriching, and detecting target molecules in a sample. These techniques include chromatography, (e.g., paper, liquid, such as high performance liquid phase (HPLC)); electrophoresis (e.g., capillary and slab electrophoresis, such as agarose or polyacrylamide gel electrophoresis (PAGE), affinity electrophoresis); affinity purification (e.g., immunoaffinity column); nucleic acid hybridization (e.g., Southern and Northern hybridizations, nucleic acid arrays); and antibody based methods (e.g., Western hybridization, antibody arrays).
Gel electrophoresis is one of the best known methods for separating, purifying and characterizing charged molecules, particularly macromolecules such as proteins or nucleic acids (Freifelder, ed., Physical Biochemistry, 2nd Ed., W.H. Freeman and Company, San Francisco (1982), pp. 276-310), In electrophoretic separations, charged molecules migrate through a supporting medium under the influence of an electric field. The supporting medium acts to suppress convection and diffusion and, in some circumstances, can act as a sieve. Electrophoresis can be used to separate molecules based on size, charge, conformation or combinations of these properties.
Most frequently, electrophoresis is carried out using a constant voltage applied across two fixed electrodes located at opposite ends of a gel medium, which results in a linear constant voltage gradient of fixed orientation. However, for very large DNA molecules (i.e., 30-2000 kb), the polymeric chain orients with the field and snakes through the gel, rendering the sieving action of the electrophoretic medium ineffective. In order to separate large DNA molecules, “field inversion” electrophoresis, in which the field orientation is reversed cyclically (see, e.g., Cane et al. (1986), Science 232:65-68), and ‘pulsed field” electrophoresis (see, e.g., Schwartz et al. (1984), Cell 37:67), in which the field is reoriented at oblique angles cyclically, have been developed. Other approaches that included alternating or varied electric field include transverse alternating field electrophoresis (TAFE) and contour-clamped homogeneous electric field (CHEF) electrophoresis (see, e.g., Gardiner et al. (1986), Somatic Cell Molec. Genet. 12:185-195; Chu et al. (1986), Science 234:1582-1585; U.S. Pat. No. 5,549,796).
In affinity electrophoresis, the support medium (e.g., gel) contains a binding partner that interacts specifically or nonspecifically with one or more desired target molecules and aids in the separation of target molecules from non-target molecules during electrophoretic migration. For example, affinity electrophoresis has been used to measure the binding affinity of proteins (Horcjsi et al. (1974), Biochim. Biophys. Ada 3 36:338-343; Chu et al. (1992), J˜Med. Chem. 35:2915-2917). In addition, vinyl-adenine-modified polyacrylamide electrophoretic media have been used to enhance the resolution of nucleic acids in capillary electrophoresis (Baba et al. (1992), Analyt. Chem. 64:1920-1924).
PCT Intl. Pub. No. WO 98/51823 describes methods of detecting target molecules using electrophoresis media containing immobilized polynucleotides as the binding for the target molecule. The target molecules are typically nucleic acids, but also can include other molecules that bind to nucleic acids, such as DNA-binding proteins and aptamer binding partners.
PCT Intl. Pub. No. WO 99/45374 describes an affinity electrophoresis process in which the direction of electrophoresis is varied in a cyclical manner, while synchronously changing one or more properties of the electrophoretic medium between two states, which alternatively favor and disfavor specific reversible binding of target molecules to the binding partners that are immobilized within the medium.
PCT Intl. Pub. No. WO 00/50644 describes methods for purifying DNA using binding partners immobilized within an electrophoretic medium. In some embodiments, the electric field is increased in strength to release target molecules that have bound to the binding partners, and in some embodiments, the direction of the electric field is reversed to remove the released target molecules for collection.
Samples that are extremely dilute with respect to the target molecules or in which the target molecule is rare, or samples that are extremely heterogeneous with respect to highly similar non-target molecules, pose particular problems of separation and detection. For example, human stool samples examined for the diagnosis of colon cancer contain large amounts of bacterial DNA and protein relative to human DNA and protein, and large amounts of normal human DNA and protein relative to, for example, a DNA or protein that is indicative of a cancer-associated mutation. Similarly, human blood samples examined for the presence of pathogenic infections contain large amounts of human DNA and proteins relative to any pathogen-derived DNA or proteins.
Similarly, environmental (e.g., watershed) or industrial (e.g., food processing) samples examined for the presence of pathogens are extremely dilute with respect to any pathogen-derived DNA or proteins. Moreover, target and non-target biomolecules that contain only slight structural differences, for example, a point mutation in a protein or nucleic acid, cannot be easily separated from normal molecules by standard electrophoretic techniques.
A need therefore remains for improved methods for the separation, isolation, enrichment and detection of target molecules in dilute or heterogeneous test samples.